Tissue culture and rapid propagation method of salvia aureocauda
Through the rapid reproduction method of tissue culture, hormones are used to regulate growth, the problem of low natural reproduction rate of yellow sage is solved, efficient tissue culture seedling induction and rooting rate is achieved, and large-scale plant production is promoted.
Patent Information
- Application Number
- CN202510313455.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-06-27
AI Technical Summary
The natural reproduction rate of yellow sage is low, making it difficult to achieve large-scale production.
The rapid breeding method of tissue culture was used to disinfect the tender stems of yellow sage and cut them into stem segments with 2 to 3 leaf axillas, and inoculate them on solid culture medium containing nutrients for primary generation, successive generation and rooting cultivation, and use hormones to regulate plant growth.
The induction rate, value-added coefficient and rooting rate of yellow sage tissue culture seedlings have been improved, and there is good application prospect.
Smart Images
Figure CN120202938A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant rapid propagation, and particularly relates to a tissue culture rapid propagation method for Salvia flava. Background Art
[0002] Salvia flava (Salvia Forrest ex Diels) is a perennial herb of the genus Salvia in the family Lamiaceae. The stem is erect, 20 - 50 cm high, solitary, and unbranched. The leaves are ovate or triangular - ovate. The verticillaster usually has 4 flowers, and the corolla is yellow, up to 2.3 - 3 cm long. The flowering period is in July, and it has an alias of yellow salvia root. It grows in forests and on hillside grasslands at an altitude of 2800 - 4200 m. According to the medicinal parts and effects, Salvia flava can be classified into the salvia group. Its rhizome is used as traditional Chinese medicine salvia root. The content of diterpenoid quinone components is high. Salvia - type Chinese herbal medicines generally contain diterpenoid quinone components such as tanshinone, and have the effects of promoting blood circulation to remove blood stasis, antibacterial and anti - inflammatory, etc.
[0003] The natural propagation rate of Salvia flava is low, so tissue culture rapid propagation technology can be used to solve this problem, which lays a foundation for effectively promoting the large - scale production of Salvia flava plants and fully exploring Salvia flava resources. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a tissue culture rapid propagation method for Salvia flava. The tissue - cultured seedlings of Salvia flava obtained by this method have high induction rate, multiplication coefficient and rooting rate, and have good application prospects.
[0005] To solve the above - mentioned technical problem, the technical scheme adopted by the present invention is as follows:
[0006] A tissue culture rapid propagation method for Salvia flava, comprising the following steps:
[0007] 1) After disinfecting the tender stems of Salvia flava, cut them into stem segments with 2 to 3 leaf axils, and sterilize them for later use;
[0008] 2) Inoculate the sterilized stem segments of Salvia flava in step 1) on the surface of a solid medium containing nutrients for primary culture; sub - culture the successfully primary - cultured sterile seedlings on the original medium;
[0009] 3) Transfer the successfully sub - cultured bud seedlings in step 2) into a 1 / 2MS or WPM medium containing hormones for rooting culture.
[0010] Furthermore, the disinfection method in step 1) is: after rinsing the stem segments of Salvia flava with distilled water, soak them in 0.1% mercuric chloride, 84 disinfectant and 7% hydrogen peroxide for 4 min respectively, and finally rinse them 3 times with sterile water.
[0011] Further, the primary medium containing nutrient components in step 2) is WPM + 0.05 mg / L - 0.2 mg / L NAA + 0.2 mg / L - 1.0 mg / L 6-BA.
[0012] Further, the primary medium containing nutrient components is WPM + 0.1 mg / L NAA + 0.2 mg / L 6-BA.
[0013] Further, in step 2), 2% sucrose and 0.5% agar are added to the primary medium, and the pH is controlled to be 5.6 - 6.0.
[0014] Further, in step 2), after 10 days of primary culture, the base of the stem segment explants inoculated on the medium begins to swell. After 25 days, the aseptic seedlings successfully cultured in the primary stage are subcultured on the original medium, subcultured once every 25 days, and continuously subcultured 3 times.
[0015] Further, the 1 / 2MS or WPM medium containing hormones in step 3) is 1 / 2MS + NAA 0.05 mg / L - 0.3 mg / L, 1 / 2MS + IBA 0.05 mg / L, WPM, WPM + NAA 0.05 mg / L - 0.3 mg / L, WPM + IBA 0.05 mg / L.
[0016] Further, the hormone-containing medium is WPM + NAA 0.1 mg / L.
[0017] Further, in step 3), 2% sucrose and 0.5% agar are added to the rooting medium.
[0018] Further, the culture conditions throughout the process are: the culture temperature is 25 - 28 °C, the light duration is 12 h / d, and the light intensity is 3000 lx.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] In the tissue culture rapid propagation method of Salvia flava provided by the present invention, hormones are important factors regulating plant growth in the tissue culture of Salvia flava. In tissue culture, rapid propagation of test-tube seedlings and regeneration, the continuous supply of hormones is mainly relied on, and a suitable ratio of cytokinin and auxin can induce the differentiation of buds and roots. In the differentiation of Salvia flava buds, using cytokinin 6-BA in combination with an appropriate concentration of NAA, the medium of WPM + 0.1 mg / L NAA + 0.2 mg / L 6-BA has the best effect, is easy to form adventitious buds, and has good growth conditions. In terms of rooting, the medium of WPM + NAA 0.05 mg / L has the best effect. Description of the Drawings
[0021] Figure 1This is the figure of the primary culture result of inducing new buds from the axils of Salvia flava stems in the present invention;
[0022] Figure 2 This is the figure of the primary culture result of growing the new buds of Salvia flava into small seedlings in the present invention;
[0023] Figure 3 This is the figure of the rooting culture result of Salvia flava in the present invention. Detailed implementation manners
[0024] The following further clarifies the present invention in conjunction with specific embodiments. The embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.
[0025] Embodiment 1
[0026] 1. Analysis of the disinfection effects of different disinfectants on explants
[0027] Take the tender stems of Salvia flava. After disinfection, cut them into stem segments with 2 to 3 axils. After rinsing with distilled water (for more than 15 minutes), take them out and soak them in 0.1% mercuric chloride, 84 disinfectant, and 7% hydrogen peroxide for 4 minutes respectively on the ultra-clean workbench. Finally, rinse them 3 times with sterile water, blot them dry with filter paper, and inoculate them on the medium of WPM + NAA 0.1 mg / L + BA 0.5 mg / L. Observe the disinfection effect after one week.
[0028] The results are shown in Table 1. The disinfection effect of 0.1% mercuric chloride is the best, and the contamination rate is zero; the disinfection effects of 84 disinfectant and 7% hydrogen peroxide are relatively poor, and the contamination rates reach 26% and 100% respectively.
[0029] Table 1 Disinfection effects of different disinfectants on explants
[0030]
[0031] 2. Effects of different hormone combinations on bud differentiation of explants
[0032] Cut the sterilized Salvia flava into stem segments with 2 to 3 axils on a sterile culture dish and inoculate them on the surface of the solid medium containing different nutrient components.
[0033] Add 3% sucrose and 0.5% agar to each medium, and adjust the pH value to 5.6 - 6.0. In each of the 10 bottles, 2 stem segments are used for primary culture in the medium. After about 10 days, the base of the explant begins to swell to produce callus, the apical bud and lateral buds begin to grow, and the stem segments thicken; after 25 days, select the sterile seedlings obtained from the primary culture and perform subculture on the original medium; subculture once every 25 days for 3 consecutive times. Investigate the number of buds and the bud length of each treatment during each subculture.
[0034] The results are shown in Table 2. On the culture media with 9 different formulations, 100% of the stem segments can induce new buds from the leaf axils ( Figure 1 ), but there are obvious differences in the number of new buds induced by the culture media with different formulations. The culture medium with the formulation of WPM + 0.2 mg / L NAA + 1.0 mg / L 6-BA induces the largest number of new buds. On average, each stem segment can induce 3 new buds. The culture medium with the formulation of WPM + 0.1 mg / L NAA + 0.5 mg / L 6-BA induces the fewest new buds. On average, each stem segment can induce 1.67 new buds. Two to three weeks after inoculation, the new buds can be seen to gradually elongate. Four to six weeks later, the new buds grow into small seedlings ( Figure 2 ). With the increase of the 6-BA concentration, the bud differentiation rate increases, but the growth potential weakens. NAA has no significant effect on proliferation and elongation within a certain range, but too high a concentration will inhibit the proliferation and elongation of the tender buds. Considering the actual situation, the effect of WPM + 0.1 mg / L NAA + 0.2 mg / L 6-BA is the best from the perspective of actual production.
[0035] Table 2 Effects of different hormone combinations on bud differentiation of explants
[0036]
[0037] 3. Effects of different hormone combinations on rooting
[0038] Select the bud seedlings about 2 cm high and with strong growth that have been successfully subcultured and transfer them to the 1 / 2MS and WPM rooting culture media treated with 7 different hormone concentrations. Add 2% sucrose and 0.5% agar to the culture media. Inoculate 30 plants for each treatment and culture them. After 2 to 3 weeks, investigate the number of rooted seedlings and the rooting rate of each treatment.
[0039] The results are shown in Table 3. The combination with WPM as the basic culture medium + various growth regulators generally has a higher final rooting rate than the combination with 1 / 2MS as the basic culture medium + various growth regulators. Among them, the rooting efficiency is the best on the WPM + NAA 0.1 mg / L culture medium, and the rooting frequency reaches 93.30%. Two weeks after inoculation into the rooting culture medium, the tissue culture seedlings can be seen to root ( Figure 3 ).
[0040] Table 3 Effects of different hormone combinations on rooting
[0041]
[0042] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A tissue culture and rapid propagation method for Salvia serrata, characterized in that: The following steps are involved: 1) Disinfect the tender stems of Sage serrata and cut them into stem segments with 2 to 3 leaf axils. Sterilize them and set aside for later use. 2) inoculating the sterilized Salvia officinalis stem segments in step 1) on the surface of a solid culture medium containing nutrients for primary culture; and subculturing the sterile seedlings successfully cultured in the primary culture medium; 3) The seedlings that have been successfully subcultured in step 2) are transferred into 1 / 2MS or WPM medium containing hormones for rooting culture.
2. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: The disinfection method in step 1) is: after washing the stem segments of Salvia officinalis with distilled water, soak them in 0.1% mercuric chloride, 84 disinfectant and 7% hydrogen peroxide for 4 minutes respectively, and finally wash them with sterile water for 3 times.
3. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: The primary culture medium containing nutrients in step 2) is WPM+0.05mg / L-0.2mg / LNAA+0.2mg / L-1.0mg / L6-BA.
4. The tissue culture and rapid propagation method of Salvia lutea according to claim 3, characterized in that: The primary culture medium containing nutrients is WPM+0.1 mg / L NAA+0.2 mg / L 6-BA.
5. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: In step 2), 3% sucrose and 0.5% agar were added to the primary culture medium, and the pH was controlled at 5.6-6.
0.
6. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: In step 2), after 10 days of primary culture, the base of the stem segment explants inoculated on the culture medium begins to swell, and after 25 days, the sterile seedlings successfully cultured in the primary culture are subcultured on the original culture medium, subcultured once every 25 days, and subcultured three times in succession.
7. The tissue culture and rapid propagation method of Salvia officinalis according to claim 1, characterized in that: The 1 / 2MS or WPM culture medium containing hormones in step 3) is 1 / 2MS+NAA0.05mg / L-0.3mg / L, 1 / 2MS+IBA0.05mg / L, WPM, WPM+NAA0.05mg / L-0.3mg / L, WPM+IBA0.05mg / L.
8. The tissue culture and rapid propagation method of Salvia lutea according to claim 7, characterized in that: The hormone-containing culture medium is WPM+NAA0.1 mg / L.
9. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: In step 3), 2% sucrose and 0.5% agar were added to the rooting medium.
10. The tissue culture and rapid propagation method of Salvia lutea according to claim 1, characterized in that: The culture conditions throughout the process are: culture temperature is 25-28°C, light hours are 12h / d, and light intensity is 30001x.